Dextran-based polymer tissue adhesive for medical use

ABSTRACT

A tissue adhesive formed by reacting an aminodextran containing primary amine groups with an oxidized dextran containing aldehyde groups is described. The dextran-based polymer tissue adhesive is particularly useful in medical applications where low swell and slow degradation are needed, for example sealing the dura, ophthalmic procedures, tissue repair, antiadhesive applications, drug delivery, and as a plug to seal a fistula or the punctum.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 from U.S.Provisional Application Ser. No. 61/003,060, filed Nov. 14, 2007.

FIELD OF THE INVENTION

The invention relates to the field of medical adhesives. Morespecifically, the invention relates to a polymer tissue adhesive formedby reacting an aminodextran containing primary amine groups with anoxidized dextran containing aldehyde groups.

BACKGROUND OF THE INVENTION

Tissue adhesives have many potential medical applications, includingwound closure, supplementing or replacing sutures or staples in internalsurgical procedures, adhesion of synthetic onlays or inlays to thecornea, drug delivery devices, and as anti-adhesion barriers to preventpost-surgical adhesions. Conventional tissue adhesives are generally notsuitable for a wide range of adhesive applications. For example,cyanoacrylate-based adhesives have been used for topical wound closure,but the release of toxic degradation products limits their use forinternal applications. Fibrin-based adhesives are slow curing, have poormechanical strength, and pose a risk of viral infection. Additionally,the Fibrin-based adhesives do not covalently bind to the underlyingtissue.

Several types of hydrogel tissue adhesives have been developed whichhave improved adhesive and cohesive properties and are nontoxic. Thesehydrogels are generally formed by reacting a component havingnucleophilic groups with a component having electrophilic groups capableof reacting with the nucleophilic groups of the first component to forma crosslinked network via covalent bonding. However, these hydrogelstypically swell or dissolve away too quickly, or lack sufficientadhesion or mechanical strength, thereby decreasing their effectivenessas surgical adhesives.

Kodokian et al. (copending and commonly owned U.S. Patent ApplicationPublication No. 2006/0078536) describe hydrogel tissue adhesives formedby reacting an oxidized polysaccharide with a water-dispersible,multi-arm polyether amine. These adhesives provide improved adhesion andcohesion properties, crosslink readily at body temperature, maintaindimensional stability initially, do not degrade rapidly, and arenontoxic to cells and non-inflammatory to tissue. Hydrogel tissueadhesives with low swell and slow degradation are needed forapplications including but not limited to, sealing the dura, ophthalmicprocedures, tissue repair, antiadhesive applications, drug delivery, andsealing a fistula or the punctum.

Polysaccharide-based hydrogels are known and various uses have beendescribed, for example, use as a drug carrier (Spiro et al., WO99/01143), use as a coating on a carrier for use in diagnostic ortherapeutic methods (Kirakossian et al., U.S. Pat. No. 7,179,660), anduse as a coating for prohibiting post surgical adhesions (Yeo et al.,U.S. Patent Application Publication No. 2008/0069857). Additionally, apolysaccharide-based hydrogel formed by reacting oxidized dextran andchitosan for use as a tissue adhesive is described by Goldmann (U.S.Patent Application Publication No. 2005/0002893) and Odermatt et al.(U.S. Patent Application Publication No. 2006/0292030).

Consequently, the problem to be solved is to provide a tissue adhesivematerial with low swell and a slow degradation rate for use in surgicalprocedures as well as other medical applications. The stated problem isaddressed herein by the discovery that hydrogels formed by the reactionof an aminodextran containing primary amine groups and an oxidizeddextran containing aldehyde groups possess these desired properties.

SUMMARY OF THE INVENTION

An embodiment provides a kit comprising:

-   -   a) at least one dextran that has been derivatized to provide at        least one aminodextran that contains primary amine groups, said        at least one dextran having a weight-average molecular weight of        about 1,000 to about 1,000,000 Daltons, said at least one        aminodextran having an amine substitution level of about 5% to        about 65%; and    -   b) at least one dextran that has been oxidized to provide at        least one oxidized dextran containing aldehyde groups, said at        least one dextran having a weight-average molecular weight of        about 1,000 to about 1,000,000 Daltons, said at least one        oxidized dextran having an equivalent weight per aldehyde group        of about 65 to about 1500 Daltons;        wherein the at least one aminodextran and the at least one        oxidized dextran are unreacted.

Another embodiment provides the kit, wherein the aminodextran is a firstaqueous solution or dispersion and the oxidized dextran a second aqueoussolution or dispersion.

Another embodiment provides the, wherein the first aqueous solution ordispersion contains the aminodextran at a concentration of about 5% toabout 70% by weight relative to the total weight of the solution ordispersion.

Another embodiment provides the kit, wherein the second aqueous solutionor dispersion contains the oxidized dextran at a concentration of about5% to about 50% by weight relative to the total weight of the solutionor dispersion.

In yet another embodiment, the kit wherein the aminodextran and theoxidized dextran are finely divided powders.

In yet another embodiment, the kit wherein the equivalent weight peraldehyde group of the oxidized dextran is about 90 to about 1500Daltons.

An embodiment provides a dried hydrogel product formed by a processcomprising the steps of:

-   -   a) reacting in a solvent (i) at least one dextran that has been        derivatized to provide at least one aminodextran that contains        primary amine groups, said at least one dextran having a        weight-average molecular weight of about 1,000 to about        1,000,000 Daltons, said at least one aminodextran having an        amine substitution level of about 5% to about 65%; with (ii) at        least one dextran that has been oxidized to provide at least one        oxidized dextran containing aldehyde groups, said at least one        dextran having a weight-average molecular weight of about 1,000        to about 1,000,000 Daltons, said at least one oxidized dextran        having an equivalent weight per aldehyde group of about 65 to        about 1500 Daltons, to form a hydrogel;    -   b) treating said hydrogel to remove at least a portion of said        solvent to form the dried hydrogel.

Another embodiment provides the dried hydrogel, wherein said driedhydrogel is a film.

Another embodiment provides the dried hydrogel, wherein the processfurther comprises comminuting the dried hydrogel to form finely dividedparticles.

Another embodiment provides the dried hydrogel, wherein the equivalentweight per aldehyde group of the oxidized dextran is about 90 to about1500 Daltons.

An embodiment provides a combination for use in coating an anatomicalsite comprising:

-   -   a) at least one dextran that has been derivatized to provide at        least one aminodextran that contains primary amine groups, said        at least one dextran having a weight-average molecular weight of        about 1,000 to about 1,000,000 Daltons, said at least one        aminodextran having an amine substitution level of about 5% to        about 65%; followed by    -   b) at least one dextran that has been oxidized to provide at        least one oxidized dextran containing aldehyde groups, said at        least one dextran having a weight-average molecular weight of        about 1,000 to about 1,000,000 Daltons, said at least one        oxidized dextran having an equivalent weight per aldehyde group        of about 65 to about 1500 Daltons; or alternatively (b) followed        by (a).

Another embodiment provides the combination, wherein the aminodextran isa first aqueous solution or dispersion and the oxidized dextran is asecond aqueous solution or dispersion.

Another embodiment provides the combination, wherein the first aqueoussolution or dispersion contains the aminodextran at a concentration ofabout 5% to about 70% by weight relative to a total weight of thesolution or dispersion.

In yet another embodiment, the combination, wherein the second aqueoussolution or dispersion contains the oxidized dextran at a concentrationof about 5% to about 50% by weight relative to the total weight of thesolution or dispersion.

In yet another embodiment, the combination, wherein the aminodextran andthe oxidized dextran are finely divided powders.

Another embodiment provides the combination, wherein the equivalentweight per aldehyde group of the oxidized dextran is about 90 to about1500 Daltons.

An embodiment provides a method for applying a coating to an anatomicalsite on tissue of a living organism comprising:

-   -   applying to the site        -   a) at least one dextran that has been derivatized to provide            at least one aminodextran that contains primary amine            groups, said at least one dextran having a weight-average            molecular weight of about 1,000 to about 1,000,000 Daltons,            said at least one aminodextran having an amine substitution            level of about 5% to about 65%; followed by        -   b) at least one dextran that has been oxidized to provide at            least one oxidized dextran containing aldehyde groups, said            at least one dextran having a weight-average molecular            weight of about 1,000 to about 1,000,000 Daltons, said at            least one oxidized dextran having an equivalent weight per            aldehyde group of about 65 to about 1500 Daltons; or            applying (b) followed by (a) and mixing (a) and (b) on the            site; or premixing (a) and (b) and applying the resulting            mixture to the site.

Another embodiment provides the method, wherein the aminodextran is inthe form of a first aqueous solution or dispersion and the oxidizeddextran is in the form of a second aqueous solution or dispersion.

Another embodiment provides the method, wherein the first aqueoussolution or dispersion contains the aminodextran at a concentration ofabout 5% to about 70% by weight relative to the total weight of thesolution or dispersion.

In yet another embodiment, the method, wherein the second aqueoussolution or dispersion contains the oxidized dextran at a concentrationof about 5% to about 50% by weight relative to the total weight of thesolution or dispersion.

In yet another embodiment, the method, wherein the aminodextran and theoxidized dextran are in the form of finely divided powders.

In yet another embodiment, the method, wherein the equivalent weight peraldehyde group of the oxidized dextran is about 90 to about 1500Daltons.

An embodiment provides a combination for use in bonding at least twoanatomical sites together, wherein the combination is applied to atleast one of the at least two anatomical sites, the combinationcomprising;

-   -   a) at least one dextran that has been derivatized to provide at        least one aminodextran that contains primary amine groups, said        at least one dextran having a weight-average molecular weight of        about 1,000 to about 1,000,000 Daltons, said at least one        aminodextran having an amine substitution level of about 5% to        about 65%; followed by    -   b) at least one dextran that has been oxidized to provide at        least one oxidized dextran containing aldehyde groups, said at        least one dextran having a weight-average molecular weight of        about 1,000 to about 1,000,000 Daltons, said at least one        oxidized dextran having an equivalent weight per aldehyde group        of about 65 to about 1500 Daltons; or alternatively (b) followed        by (a).

Another embodiment provides the combination, wherein the aminodextran isa first aqueous solution or dispersion and the oxidized dextran is asecond aqueous solution or dispersion.

Another embodiment provides the combination, wherein the first aqueoussolution or dispersion contains the aminodextran at a concentration ofabout 5% to about 70% by weight relative to a total weight of thesolution or dispersion.

In yet another embodiment, the combination, wherein the second aqueoussolution or dispersion contains the oxidized dextran at a concentrationof about 5% to about 50% by weight relative to the total weight of thesolution or dispersion.

In yet another embodiment, the combination, wherein the at least oneaminodextran and the at least one oxidized dextran are finely dividedpowders.

In yet another embodiment, the combination, wherein the equivalentweight per aldehyde group of the oxidized dextran is about 90 to about1500 Daltons.

Another embodiment provides a product for use in a coating an anatomicalsite comprising the dried hydrogel.

Another embodiment provides the product, wherein said dried hydrogel isa film.

Another embodiment provides the product, wherein said dried hydrogel isfinely divided particles.

An embodiment provides a method for bonding at least two anatomicalsites together comprising:

-   -   applying to at least one of the at least two anatomical sites:        -   a) at least one dextran that has been derivatized to provide            at least one aminodextran that contains primary amine            groups, said at least one dextran having a weight-average            molecular weight of about 1,000 to about 1,000,000 Daltons,            said at least one aminodextran having an amine substitution            level of about 5% to about 65%; followed by        -   b) at least one dextran that has been oxidized to provide at            least one oxidized dextran containing aldehyde groups, said            at least one dextran having a weight-average molecular            weight of about 1,000 to about 1,000,000 Daltons, said at            least one oxidized dextran having an equivalent weight per            aldehyde group of about 65 to about 1500 Daltons; or            alternatively, applying (b) followed by (a) and mixing (a)            and (b) on the at least one site, or alternatively,            premixing (a) and (b) and applying the resulting mixture to            the at least one site; and contacting the at least two            anatomical sites together.

Another embodiment provides the method, wherein the aminodextran is inthe form of a first aqueous solution or dispersion and the oxidizeddextran is in the form of a second aqueous solution or dispersion.

Another embodiment provides the method, wherein the first aqueoussolution or dispersion contains the aminodextran at a concentration ofabout 5% to about 70% by weight relative to the total weight of thesolution or dispersion.

In yet another embodiment, the method wherein the second aqueoussolution or dispersion contains the oxidized dextran at a concentrationof about 5% to about 50% by weight relative to the total weight of thesolution or dispersion.

In yet another embodiment, the method wherein the at least oneaminodextran and the at least one oxidized dextran are in the form offinely divided powders.

Another embodiment provides the method, wherein the equivalent weightper aldehyde group of the oxidized dextran is about 90 to about 1500Daltons.

In yet another embodiment, a method for applying a coating to ananatomical site on tissue of a living organism comprising applying tothe site the dried hydrogel.

In yet another embodiment, the method, wherein said dried hydrogel is inthe form of a film.

In yet another embodiment, the method, wherein said dried hydrogel is inthe form of finely divided particles.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a polymer tissue adhesive formed by reacting atleast one aminodextran containing primary amine groups and at least oneoxidized dextran containing aldehyde groups. The dextran-based polymertissue adhesive is particularly useful in medical applications where lowswell and slow degradation are needed, for example sealing the dura,ophthalmic procedures, tissue repair, antiadhesive applications, drugdelivery, and as a plug to seal a fistula or the punctum. Due to thepositive charge on the aminodextran, the polymer tissue adhesivedisclosed herein may possess antimicrobial properties and promote woundhealing.

The following definitions are used herein and should be referred to forinterpretation of the claims and the specification.

The term “oxidized dextran” refers to dextran that has been reacted withan oxidizing agent to introduce aldehyde groups into the molecule.

The terms “aminodextran” and “dextran amine” are used interchangeablyherein to refer to dextran that has been derivatized (i.e., chemicallymodified) to contain primary amine groups.

The term “equivalent weight per aldehyde group” refers to the averagemolecular weight of the compound divided by the number of aldehydegroups in the molecule.

The term “amine substitution level” as used herein, refers to thepercent of saccharide rings in dextran that are substituted with aprimary amine group. The amine substitution level is determined usingproton nuclear magnetic resonance (NMR) spectroscopy, as describedherein.

The term “hydrogel” refers to a water-swellable polymeric matrix,consisting of a three-dimensional network of macromolecules heldtogether by covalent or non-covalent crosslinks, that can absorb asubstantial amount of water to form an elastic gel.

The term “crosslink” refers to a bond or chain of atoms attached betweenand linking two different polymer chains.

The term “dried hydrogel” refers to a hydrogel that has been treated toremove at least a portion of the solvent contained therein. Preferably,substantially all of the solvent is removed from the hydrogel.

The term “% by weight” as used herein refers to the weight percentrelative to the total weight of the solution or dispersion, unlessotherwise specified.

The term “anatomical site” refers to any external or internal part ofthe body of humans or animals.

The term “tissue” refers to any tissue, both living and dead, in humansor animals.

The term medical application is refers to medical applications asrelated to humans and animals.

The meaning of abbreviations used is as follows: “min” means minute(s),“h” means hour(s), “sec” means second(s), “d” means day(s), “mL” meansmilliliter(s), “L” means liter(s), “μL” means microliter(s), “cm” meanscentimeter(s), “mm” means millimeter(s), “μm” means micrometer(s), “mol”means mole(s), “mmol” means millimole(s), “g” means gram(s), “mg” meansmilligram(s), “wt %” means percent by weight, “mol %” means molepercent, “M” means molar concentration, “Vol” means volume, “v/v” meansvolume per volume, “Da” means Daltons, “kDa” means kiloDaltons, “mw”means molecular weight, “MWCO” means molecular weight cut-off, “kPa”means kilopascals, “¹H NMR” means proton nuclear magnetic resonancespectroscopy, “ppm” means parts per million, “PBS” meansphosphate-buffered saline.

Dextrans

Dextran is a complex, branched polysaccharide that includes many glucosemoieties joined together via glycosidic linkages to form straightchains. Dextrans having various average molecular weights are availablefrom commercial sources such as Sigma-Aldrich (Milwaukee, Wis.) andPharmacosmos A/S (Holbaek, Denmark). Typically, commercial preparationsof dextran are a heterogeneous mixture having a distribution ofdifferent molecular weights, as well as a variable degree of branching,and are characterized by various molecular weight averages, for example,the weight-average molecular weight, or the number-average molecularweight, as is known in the art. Suitable dextrans have a weight-averagemolecular weight of about 1,000 to about 1,000,000 Daltons, preferablyfrom about 3,000 to about 250,000 Daltons.

Oxidized Dextran Containing Aldehyde Groups

One reactant used to prepare the polymer tissue adhesive disclosedherein is an oxidized dextran containing aldehyde groups. Oxidizeddextran may be prepared by oxidizing dextran using any suitableoxidizing agent, including but not limited to, periodates,hypochlorites, ozone, peroxides, hydroperoxides, persulfates, andpercarbonates. In one embodiment, the dextran is oxidized by reactionwith sodium periodate, for example as described by Mo et al. (J.Biomater. Sci. Polymer Edn. 11:341-351, 2000). The dextran may bereacted with different amounts of periodate to give dextrans withdifferent degrees of oxidation and therefore, different amounts ofaldehyde groups, as described in detail in the General Methods Sectionof the Examples herein. The aldehyde content of the oxidized dextran maybe determined using methods known in the art. For example, thedialdehyde content of the oxidized dextran may be determined using themethod described by Hofreiter et al. (Anal Chem. 27:1930-1931, 1955), asdescribed in detail in the General Methods Section of the Examplesherein. In that method, the amount of alkali consumed per mole ofdialdehyde in the oxidized dextran, under specific reaction conditions,is determined by a pH titration. The equivalent weight per aldehydegroup of the oxidized dextran is about 65 to about 1500 Daltons, inaddition about 90 to about 1500 Daltons.

In one embodiment, the oxidized dextran is prepared from dextran havinga weight-average molecular weight of 8,500 to 11,500 Dalton, and has anequivalent weight per aldehyde group of about 146 Daltons.

In another embodiment, the oxidized dextran is prepared from dextranhaving a weight-average molecular weight of 8,500 to 11,500 Daltons andhas an equivalent weight per aldehyde group of about 109 Daltons.

Aminodextran Containing Primary Amine Groups

The second reactant used to prepare the polymer tissue adhesivedisclosed herein is aminodextran containing primary amine groups.Aminodextran containing primary amine groups can be prepared by chemicalderivatization of dextran using methods known in the art. For example,dextran can be oxidized to produce oxidized dextran containing aldehydegroups, as described above. Then, the oxidized dextran can be reactedwith a diamine, such as hexamethylene diamine, ethylene diamine,propylene diamine, and the like, to form Schiff base linkages.Optionally, the Schiff base linkages may be treated with a reducingagent such as sodium borohydride to form stable carbon-nitrogen bonds,as described in detail in the Examples herein. Aminodextran may also beprepared by reacting dextran with cyanogen bromide, followed by reactionwith a diamine. Additionally, aminodextran can be prepared by themethods described by Kirakossian et al. (U.S. Pat. No. 7,179,660,Example A). The amine substitution level of the aminodextran isdetermined using proton NMR by determining the ratio of the integral ofthe peaks corresponding to the pendant amine-containing groups to thesum of the integrals of the peaks corresponding to the anomeric protonsof the glucose ring and comparing it to the expected ratio for a fullyderivatized product, as shown in the Examples herein.

In one embodiment, the amine substitution level of the aminodextran isfrom about 5% to about 65%.

In another embodiment, the aminodextran is prepared from dextran havinga weight-average molecular weight of 8,500 to 11,500 Daltons and has anamine substitution level of about 46%.

In another embodiment, the aminodextran is prepared from dextran havinga weight-average molecular weight of 60,000 to 90,000 Daltons and has anamine substitution level of about 32%.

In another embodiment, the aminodextran is prepared from dextran havinga weight-average molecular weight of 400 to 500 kiloDaltons and has anamine substitution level of about 38%.

In another embodiment, the aminodextran is prepared from dextran havinga weight-average molecular weight of about 2,000 kiloDaltons and has anamine substitution level of about 36%.

Methods of Using Dextran-Based Polymer Tissue Adhesive

The dextran-based polymer tissue adhesive disclosed herein may be usedin various forms. In one embodiment, the oxidized dextran containingaldehyde groups and the aminodextran containing primary amine groups areused in the form of aqueous solutions or dispersions. Dispersion, asused herein, refers to a colloidal suspension capable of reacting with asecond reactant in an aqueous medium. To prepare an aqueous solution ordispersion comprising aminodextran containing primary amine groups(referred to herein as the “first aqueous solution or dispersion”), atleast one aminodextran is added to water to give a concentration ofabout 5% to about 70% by weight, in addition about 5% to about 50% byweight, in addition about 10% to about 50% by weight, and in additionabout 10% to about 30% by weight, relative to the total weight of thesolution or dispersion. Mixtures of different aminodextrans, havingdifferent average molecular weights and/or different equivalent weightsper amine group, may also be used. If a mixture of differentaminodextrans is used, the total concentration of the aminodextrans isabout 5% to about 70% by weight, in addition about 5% to about 50% byweight, in addition about 10% to about 50% by weight, and in additionabout 10% to about 30% by weight Similarly, to prepare an aqueoussolution or dispersion comprising oxidized dextran containing aldehydegroups (referred to herein as the “second aqueous solution ordispersion”), at least one oxidized dextran is added to water to give aconcentration of about 5% to about 50% by weight, in addition from about10% to about 30% by weight, relative to the total weight of the solutionor dispersion. Mixtures of different oxidized dextrans containingaldehyde groups, having different average molecular weights and/ordifferent equivalent weights per aldehyde group, may also be used. If amixture of different oxidized dextrans is used, the total concentrationof the oxidized dextrans is about 5% to about 50% by weight, in additionabout 10% to about 30% by weight, relative to the total weight of thesolution or dispersion. The optimal concentrations of the two dextransolutions or dispersions to be used depends on the application, and canbe readily determined by one skilled in the art using routineexperimentation.

For use on living tissue, it is preferred that the aqueous solution ordispersion comprising the oxidized dextran(s) and the aqueous solutionor dispersion comprising the aminodextran(s) be sterilized to preventinfection. Any suitable sterilization method known in the art that doesnot adversely affect the ability of the components to react to form aneffective hydrogel may be used. For example the first aqueous solutionor dispersion comprising at least one aminodextran may be sterilizedusing heat, ethylene oxide sterilization, ultra-violet radiation, orultra-filtration through a 0.2 μm pore membrane. The second aqueoussolution or dispersion comprising at least one oxidized dextran may besterilized using heat, electron beam irradiation, gamma irradiation,ethylene oxide sterilization, ultra-violet radiation, orultra-filtration through a 0.2 μm pore membrane.

The first aqueous solution or dispersion comprising the aminodextran(s)and/or the second aqueous solution or dispersion comprising the oxidizeddextran(s) may further comprise various additives depending on theintended application. Preferably, the additive is compatible with thedextran. Specifically, the additive does not contain groups that wouldinterfere with effective gelation of the hydrogel. The amount of theadditive used depends on the particular application and may be readilydetermined by one skilled in the art using routine experimentation. Forexample, the first aqueous solution or dispersion and/or the secondaqueous solution or dispersion may comprise at least one additiveselected from pH modifiers, viscosity modifiers, antimicrobials,colorants, surfactants, pharmaceutical drugs and therapeutic agents.

The aqueous solution(s) or dispersion(s) may optionally include at leastone pH modifier to adjust the pH of the solution(s). Suitable pHmodifiers are well known in the art. The pH modifier may be an acidic orbasic compound. Examples of acidic pH modifiers include, but are notlimited to, carboxylic acids, inorganic acids, and sulfonic acids.Examples of basic pH modifiers include, but are not limited to,hydroxides, alkoxides, nitrogen-containing compounds other than primaryand secondary amines, and basic carbonates and phosphates.

The aqueous solution(s) or dispersion(s) may optionally include at leastone thickener. The thickener may be selected from among known viscositymodifiers, including, but not limited to, polysaccharides andderivatives thereof, such as starch or hydroxyethyl cellulose.

The aqueous solution(s) or dispersion(s) may optionally include at leastone antimicrobial agent. Suitable antimicrobial preservatives are wellknown in the art. Examples of suitable antimicrobials include, but arenot limited to, alkyl parabens, such as methylparaben, ethylparaben,propylparaben, and butylparaben; triclosan; chlorhexidine; cresol;chlorocresol; hydroquinone; sodium benzoate; and potassium benzoate.

The aqueous solution(s) or dispersion(s) may also optionally include atleast one colorant to enhance the visibility of the solution(s).Suitable colorants include dyes, pigments, and natural coloring agents.Examples of suitable colorants include, but are not limited to, FD&C andD&C colorants, such as FD&C Violet No. 2, FD&C Blue No. 1, D&C Green No.6, D&C Green No. 5, D&C Violet No. 2; and natural colorants such asbeetroot red, canthaxanthin, chlorophyll, eosin, saffron, and carmine.

The aqueous solution(s) or dispersion(s) may also optionally include atleast one surfactant. Surfactant, as used herein, refers to a compoundthat lowers the surface tension of water. The surfactant may be an ionicsurfactant, such as sodium lauryl sulfate, or a neutral surfactant, suchas polyoxyethylene ethers, polyoxyethylene esters, and polyoxyethylenesorbitan.

Additionally, the aqueous solution(s) or dispersion(s) may optionallyinclude at least one pharmaceutical drug or therapeutic agent. Suitabledrugs and therapeutic agents are well known in the art (for example seethe United States Pharmacopeia (USP), Physician's Desk Reference(Thomson Publishing), The Merck Manual of Diagnosis and Therapy 18thed., Mark H. Beers and Robert Berkow (eds.), Merck Publishing Group,2006; or, in the case of animals, The Merck Veterinary Manual, 9th ed.,Kahn, C.A. (ed.), Merck Publishing Group, 2005). Nonlimiting examplesinclude, but are not limited to, anti-inflammatory agents, for example,glucocorticoids such as prednisone, dexamethasone, budesonide;non-steroidal anti-inflammatory agents such as indomethacin, salicylicacid acetate, ibuprofen, sulindac, piroxicam, and naproxen; fibrinolyticagents such as a tissue plasminogen activator and streptokinase;anti-coagulants such as heparin, hirudin, ancrod, dicumarol, sincumar,iloprost, L-arginine, dipyramidole and other platelet functioninhibitors; antibodies; nucleic acids; peptides; hormones; growthfactors; cytokines; chemokines; clotting factors; endogenous clottinginhibitors; antibacterial agents; antiviral agents; antifungal agents;anti-cancer agents; cell adhesion inhibitors; healing promoters;vaccines; thrombogenic agents, such as thrombin, fibrinogen,homocysteine, and estramustine; radio-opaque compounds, such as bariumsulfate and gold particles and radiolabels.

The first aqueous solution or dispersion comprising the aminodextran(s)and the second aqueous solution or dispersion comprising the oxidizeddextran(s) may be applied to an anatomical site on tissue of a livingorganism in any number of ways. Once both solutions or dispersions areapplied to a site, they crosslink to form a hydrogel, a process referredto herein as curing, typically in about 2 seconds to about 2 minutes.

In one embodiment, the two aqueous solutions or dispersions are appliedto the site sequentially using any suitable means including, but notlimited to, spraying, brushing with a cotton swab or brush, or extrusionusing a pipette, or a syringe. The solutions or dispersions may beapplied in any order. Then, the solutions or dispersions are mixed onthe site using any suitable device, such as a cotton swab, a spatula, orthe tip of the pipette or syringe.

In another embodiment, the two aqueous solutions or dispersions aremixed manually before application to the site. The resulting mixture isthen applied to the site before it completely cures using a suitableapplicator, as described above.

In another embodiment, the two aqueous solutions or dispersions arecontained in separate barrels of a double-barrel syringe. In this waythe two aqueous solutions or dispersions are applied simultaneously tothe site with the syringe. Suitable double-barrel syringe applicatorsare known in the art. For example, Redl describes several suitableapplicators for use in the invention in U.S. Pat. No. 6,620,125,(particularly FIGS. 1, 5, and 6, which are described in Columns 4, line10 through column 6, line 47). Additionally, the double barrel syringemay contain a motionless mixer, such as that available from ConProtec,Inc. (Salem, N.H.) or Mixpac Systems AG (Rotkreuz, Switzerland), at thetip to effect mixing of the two aqueous solutions or dispersions priorto application. Alternatively, the mixing tip may be equipped with aspray head, such as that described by Cruise et al. in U.S. Pat. No.6,458,147. Additionally, the mixture of the two aqueous solutions ordispersions from the double-barrel syringe may be applied to the siteusing a catheter or endoscope. Devices for mixing a two liquid componenttissue adhesive and delivering the resulting mixture endoscopically areknown in the art and may be adapted for the mixing and delivery of thetwo aqueous solutions or dispersions disclosed herein (see for example,Nielson, U.S. Pat. No. 6,723,067; and Redl et al., U.S. Pat. No.4,631,055). Suitable delivery devices for use in ophthalmicapplications, where small volumes of the two aqueous solutions ordispersions or the mixture thereof are required, are also known in theart (see for example Miller et al., U.S. Pat. No. 4,874,368, andcopending and commonly owned U.S. Patent Application No. 61/002,071).

In another embodiment, the first aqueous solution or dispersion and thesecond aqueous solution or dispersion are applied to the sitesimultaneously where they mix to form a hydrogel. The two aqueoussolutions or dispersions may be applied to the site in various ways, forexample, using a dual-lumen catheter, such as those available fromBistech, Inc. (Woburn, Mass.). Additionally, injection devices forintroducing two liquid components endoscopically into the bodysimultaneously are known in the art and may be adapted for the deliveryof the two aqueous solutions or dispersions disclosed herein (see forexample, Linder et al., U.S. Pat. No. 5,322,510).

In another embodiment, the two aqueous solutions or dispersions may beapplied to the site using a spray device, such as those described byFukunaga et al. (U.S. Pat. No. 5,582,596), Delmotte et al. (U.S. Pat.No. 5,989,215) or Sawhney (U.S. Pat. No. 6,179,862).

In another embodiment, the two aqueous solutions or dispersions may beapplied to the site using a minimally invasive surgical applicator, suchas those described by Sawhney (U.S. Pat. No. 7,347,850).

In another embodiment, the tissue adhesive disclosed herein is used tobond at least two anatomical sites together. In this embodiment, theaqueous solution or dispersion comprising the oxidized dextran(s) isapplied to at least one anatomical site, and the aqueous solution ordispersion comprising the aminodextran(s) is applied to at least one ofeither the same site or one other site. The two or more sites arecontacted and held together manually or using some other means, such asa surgical clamp, for a time sufficient for the mixture to cure,typically from about 2 seconds to about 2 minutes. Alternatively, amixture of the two aqueous solutions or dispersions either premixedmanually or using a double-barrel syringe applicator, is applied to atleast one of the anatomical sites to be bonded. The two or more sitesare contacted and held together manually or using some other means, suchas a surgical clamp, for a time sufficient for the mixture to cure.

In another embodiment, the oxidized dextran and the aminodextran areused in the form of finely divided powders. The powders may be preparedusing any suitable method. For example, the aqueous solutions describedabove may be dried using heat, vacuum, a combination of heat and vacuum,or by lyophilization, to form powders. Optionally, the powders may becomminuted into finer particles using methods known in the artincluding, but not limited to, grinding, milling, or crushing with amortar and pestle. The finely divided powders may be sterilized usingthe methods described above. The finely divided powders may be appliedto an anatomical site on tissue of a living organism in a variety ofways. For example, the powders may be individually applied to the sitein any order by sprinkling or spraying. Additionally, the two powdersmay be premixed and the resulting mixture applied to the site using themethods described above. The powders may be hydrated on the site by theaddition of a suitable buffer (e.g., phosphate-buffered saline) or bythe physiological fluids present at the site. The finely divided powdersmay also be used to bond two anatomical sites together as describedabove for the aqueous solutions or dispersions.

In another embodiment, the dextran-based polymer tissue adhesivedisclosed herein is used in the form of a dried hydrogel. In thisembodiment, a hydrogel is prepared by mixing a solution or dispersioncomprising at least one oxidized dextran with a solution or dispersioncomprising at least one aminodextran to form a hydrogel. The solutionsor dispersions may be prepared in any suitable solvent, including butnot limited to, water, ethanol, isopropanol, tetrahydrofuran, hexanes,polyethylene glycol, and mixtures thereof. If a mixture of solvents isused, it is preferable to use solvents that are miscible with eachother. In one embodiment, the solvent is water. The solutions ordispersions may further comprise various additives depending on theintended application. Any of the additives described above may be used.The hydrogel is then treated to remove at least a portion of the solventcontained therein to form the dried hydrogel. Preferably, substantiallyall of the solvent is removed from the hydrogel. The solvent may beremoved from the hydrogel using methods known in the art, for example,using heat, vacuum, a combination of heat and vacuum, or flowing astream of dry air or a dry inert gas such as nitrogen over the hydrogel.The dried hydrogel may be sterilized using the methods described above.The dried hydrogel may be applied to an anatomical site in a number ofways, as described below. The dried hydrogel may be hydrated on the siteby the addition of a suitable buffer (e.g., phosphate-buffered saline)or by the physiological fluids present at the site.

In one embodiment, the dried hydrogel is used in the form of a film. Thedried hydrogel film may be formed by casting a mixture of the aqueoussolutions or dispersions on a suitable substrate and treating theresulting hydrogel to form a dried hydrogel film. The dried hydrogelfilm may be applied directly to an anatomical site. Additionally, thedried hydrogel film may be used to bond two anatomical sites together.

In another embodiment, the dried hydrogel is used in the form of finelydivided particles. The dried hydrogel particles may be formed bycomminuting the dried hydrogel using methods known in the art,including, but not limited to, grinding, milling, or crushing with amortar and pestle. The dried hydrogel may be applied to an anatomicalsite in a variety of ways, such as sprinkling or spraying, and may alsobe used to bond two anatomical sites together.

Kits

In one embodiment, the invention provides a kit comprising at least oneoxidized dextran containing aldehyde groups and at least oneaminodextran containing primary amine groups wherein the at least oneoxidized dextran and the at least one aminodextran are unreacted;specifically, the at least one oxidized dextran and the at least oneaminodextran are not crosslinked to form a hydrogel.

In one embodiment, the kit comprises at least one oxidized dextran andat least one aminodextran in the form of aqueous solutions ordispersions, as described above. Each of the aqueous solutions ordispersions may be contained in any suitable vessel, such as a vial or asyringe barrel.

In another embodiment, the kit comprises at least one oxidized dextranand at least one aminodextran in the form of finely divided powders, asdescribed above. The powders may be contained in separate containers orthey may be premixed and contained in a single container. The kit mayalso comprise a buffer solution for hydrating the powders.

In another embodiment, the kit comprises a dried hydrogel formed byreacting at least one oxidized dextran with at least one aminodextran,as described above. The dried hydrogel may be in the form of a film,finely divided particles, or other dried forms. The kit may furthercomprise a buffer for hydrating the dried hydrogel. The dried hydrogelparticles may be contained in any suitable container.

Medical Applications

The dextran-based polymer tissue adhesive disclosed herein isparticularly suitable for applications requiring low swell and slowdegradation, for example sealing the dura, ophthalmic procedures, tissuerepair, antiadhesive applications, drug delivery, and as a plug to seala fistula or the punctum.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and the Examples that follow, one skilled inthe art can ascertain the essential characteristics of this invention,and without departing from the spirit and scope thereof, can makevarious changes and modifications of the invention to adapt it tovarious uses and conditions.

A reference to “Aldrich” or a reference to “Sigma” means the saidchemical or ingredient was obtained from Sigma-Aldrich, St. Louis, Mo.

-   -   All water used in these Examples was distilled-deionized water        unless otherwise stated.

General Methods Preparation of Oxidized Dextran

The following procedure was used to prepare an oxidized dextran, alsoreferred to herein as dextran aldehyde, with about 50% aldehyde contentconversion from dextran having a weight-average molecular weight of8,500-11,500 Da. This dextran aldehyde is referred to herein as D10K-50.Other aldehyde conversions were obtained by varying the concentration ofthe periodate solution used. Likewise dextrans of other molecularweights were oxidized to provide the analogous oxidized dextran.Specifically, the following dextran aldehydes were prepared:weight-average molecular weight of 8.500-11,500 Da with an oxidationconversion of 50%, equivalent weight per aldehyde group of about 146 Da(D10K-50); weight-average molecular weight of 8,500-11,500 Da with anoxidation conversion of 65%, equivalent weight per aldehyde group ofabout 109 Da (D10K-65); weight-average molecular weight of 8.500-11,500Da with an oxidation conversion of 20%, equivalent weight per aldehydegroup of about 389 Da (D10K-20); weight-average molecular weight of60,000-90,000 Da with an oxidation conversion of 50%, equivalent weightper aldehyde group of about 146 Da (D60K-50); weight-average molecularweight of 400-500 kDa with an oxidation conversion of 50%, equivalentweight per aldehyde group of about 146 Da (D450K-50); and weight-averagemolecular weight of approximately 2,000 kDa with an oxidation conversionof 46%, equivalent weight per aldehyde group of about 160 Da(D2000K-46).

Dextran (19.0 g; 0.12 mol saccharide rings; weight-average molecularweight of 8,500-11,500 Da; Sigma, product number D9260) was added to 170g of water in a 500 mL round bottom flask. The mixture was stirred for15 to 30 min to produce a solution; then a solution of 17.7 g (0.083mol; mw=213.9) sodium periodate in 160 g of water was added to thedextran solution all at once. The mixture was stirred at roomtemperature for 5 h. After this time, the solution was removed from theround bottom flask, divided into four equal volumes and dispensed into 4dialysis membrane tubes (MWCO=3500 Da). The tubes were dialyzed indistilled-deionized water for 4 days, during which time the water waschanged twice daily. The aqueous solutions were removed from thedialysis tubes, placed in wide-mouth polyethylene containers and frozenusing liquid nitrogen, and lyophilized to afford white, fluffy oxidizeddextran.

The dialdehyde content in the resulting oxidized dextran was determinedusing the following procedure. The oxidized dextran (0.1250 g) was addedto 10 mL of 0.25 M NaOH in a 250 mL Erlenmeyer flask. The mixture wasgently swirled and then placed in a temperature-controlled sonicatorbath at 40° C. for 5 min until all the material dissolved, giving a darkyellow solution. The sample was removed from the bath and the flask wascooled under cold tap water for 5 min. Then 15.00 mL of 0.25 M HCl wasadded to the solution, followed by the addition of 50 mL of water and 1mL of 0.2% phenolphthalein solution. This solution was titrated with0.25 M NaOH to an endpoint determined by a color change from yellow topurple/violet. The same titration was carried out on a sample of thestarting dextran to afford a background aldehyde content. The dialdehydecontent, also referred to herein as the oxidation conversion or thedegree of oxidation, in the oxidized dextran sample was calculated usingthe following formula:

${{Dialdehyde}\mspace{14mu} {Content}} = {\frac{( {{Vb} - {Va}} )_{s}}{W_{s}/M} - {\frac{( {{Vb} - {Va}} )_{p}}{W_{p}/M} \times 100\%}}$

Vb=total meq of baseVa=total meq of acidW=dry sample weight (mg)M=weight-average molecular weight of dextran repeat unit (162)s=oxidized samplep=original samplePreparation of Aminodextran Having an Amine Substitution Level of 32%from Dextran Having a Weight-Average Molecular Weight of 60,000-90,000Da (D60K-32 Amine)

Dextran aldehyde (D60K-50) with an oxidation conversion of 50%,equivalent weight per aldehyde group of about 146 Da, was prepared asdescribed above using dextran having a weight-average molecular weightof 60,000-90,000 Da (Sigma, product number D3759). The dextran aldehyde(5.0 g) was dissolved in 500 mL of 0.1 M borate buffer, pH 11. Thedextran aldehyde solution was added slowly over 5 h using an additionfunnel to a basic solution of hexamethylene diamine in water (296 mL).The mixture was stirred at room temperature for 24 h. Then, sodiumborohydride (4.14 g) was added, and the reaction mixture was stirred for24 h. The reduction step was repeated with another addition of 4.14 g ofsodium borohydride with stirring for another 24 h. The resultingsolution was dialyzed in water using a 3500 MWCO dialysis membrane for 2days, with 2 water exchanges, then lyophilized to dryness. The resultingaminodextran is referred to herein as D60K-32 amine.

¹H NMR (D₂O): 1.40 ppm (broad, integration 2.67), 1.52 ppm (broad,integration 1.16), 1.66 ppm (broad, integration 1.35), 2.97 ppm (broad,integration 1.05), 3.51-3.92 ppm (broad multiplet, integration 9.54),4.98 ppm (broad, anomeric proton, integration 1).

The level of amine substitution was determined by ¹H NMR to be 32% bycalculating the ratio of the sum of the integrals at 1.40-1.66 ppm(NCH₂(CH₂)₄CH₂NH₂) to the integral at 4.98 (anomeric protons) andcomparing it to the expected ratio for a fully derivatized product. Forexample, for a fully derivatized product, a ratio of 16:1 is expected asthe ratio of the sum of the integrals at 1.40-1.66 ppm(NCH₂(CH₂)₄CH₂NH₂) to the integral at 4.98 (anomeric protons).

Elemental Analysis: % C 45.53, % H 7.34, % O 40.99, % N 4.46.

Preparation of Aminodextran Having an Amine Substitution Level of 46%from Dextran Having a Weight-Average Molecular Weight of 8,500-11,500Daltons (D10K-46 Amine)

Dextran aldehyde (D10K-50) with an oxidation conversion of 50%,equivalent weight per aldehyde group of about 146 Da, was prepared asdescribed above using dextran having a weight-average molecular weightof 8,500-11,500 Da. The dextran aldehyde (5.0 g) was dissolved in 500 mLof 0.1 M borate buffer, pH 11. The dextran aldehyde solution was addedslowly over 5 h using a syringe pump to a basic solution ofhexamethylene diamine (3.63 g, 0.031 mol) dissolved in 296 mL of water.The mixture was stirred at room temperature for 24 h. Then, sodiumborohydride (4.14 g, 0.10 mol) was added, and the reaction mixture wasstirred for 24 h at room temperature. The resulting solution wasdialyzed in water using a 3500 MWCO dialysis membrane for 4 days, with 4water exchanges, then lyophilized to dryness. The resulting aminodextranis referred to herein as D10K-46 amine.

¹H NMR (D₂O): 1.40 ppm, 1.54 ppm, 1.64 ppm (sum of integral of 1.40-1.64ppm: 17.36), 2.3-2.96 ppm (broad, integral 11.16), 3.57-3.93 ppm (broadmultiplet, integral 20.29), 4.98 ppm (broad, anomeric proton, integral2.41).

The level of amine substitution was determined by ¹H NMR to be 46% bycalculating the ratio of the sum of the integrals at 1.40-1.64 ppm tothe integral at 4.98 (anomeric protons) and comparing it to the expectedratio for a fully derivatized product (expected ratio of 16:1, asdescribed above).

Elemental Analysis: % C 9.42, % H 7.91, % O 35.30, % N 6.48.

Preparation of Aminodextran Having an Amine Substitution Level of 38%from Dextran Having a Weight-Average Molecular Weight of 400-500 kDa(D450K-38 Amine

Dextran aldehyde (D450K-50) with an oxidation conversion of 50%,equivalent weight per aldehyde group of about 146 Da, was prepared asdescribed above using dextran having a weight-average molecular weightof 400-500 kDa (Sigma, product number D1037). The dextran aldehyde (5.0g) was dissolved in 500 mL of 0.1 M borate buffer, pH 11. The dextranaldehyde solution was added slowly over 5 h using an addition funnel toa basic solution of hexamethylene diamine in 296 mL of water. Themixture was stirred at room temperature for 24 h. Then, sodiumborohydride (4.14 g) was added, and the reaction mixture was stirred for24 h. The reduction step was repeated with another addition of 4.14 g ofsodium borohydride with stirring for another 24 h. The resultingsolution was dialyzed in water using a 3500 MWCO dialysis membrane for 2days, with 2 water exchanges, then lyophilized to dryness. The resultingaminodextran is referred to herein as D450K-38 amine.

¹H NMR (D₂O): 1.39 ppm, 1.49 ppm, 1.66 ppm (sum of integrals of1.39-1.66 ppm is 6.09), 2.94 ppm (broad), 3.57-3.92 ppm (broad, integral9.29), 4.96 ppm (broad, anomeric proton, integral 1.0).

The level of amine substitution was determined by ¹H NMR to be 38% bycalculating the ratio of the sum of the integrals at 1.40-1.64 ppm tothe integral at 4.98 (anomeric protons) and comparing it to the expectedratio for a fully derivatized product (expected ratio of 16:1, asdescribed above).

Preparation of Aminodextran Having an Amine Substitution Level of 36%from Dextran Having a Weight-Average Molecular Weight of Approximately2,000 kDa (D2000K-36 Amine)

Dextran aldehyde (D2000K-46) with an oxidation conversion of 46%,equivalent weight per aldehyde group of about 160 Da, was prepared asdescribed above using dextran having a weight-average molecular weightof approximately 2,000 kDa (Sigma, product number D5376). The dextranaldehyde (5.0 g) was dissolved in 500 mL of 0.1 M borate buffer, pH 11.The dextran aldehyde solution was added slowly over 5 h using anaddition funnel to a basic solution of hexamethylene diamine in 296 mLof water. The mixture was stirred at room temperature for 24 h. Then,sodium borohydride (4.14 g) was added and the reaction mixture wasstirred for 24 h. The reduction step was repeated with another additionof 4.14 g of sodium borohydride with stirring for another 24 h. Theresulting solution was dialyzed in water using a 3500 MWCO dialysismembrane for 2 days, with 2 water exchanges, then lyophilized to drynessto yield 1.42 g of white solid. The resulting aminodextran is referredto herein as D2000K-36 amine.

¹H NMR (D₂O): 1.39 ppm (broad, integral 6.96), 1.51 ppm (broad, integral3.56), 1.65 ppm (broad, integral 2.26), 2.9 ppm (broad, integral 3.39),3.56-3.92 ppm (broad multiplet, integral 19.78), 4.96 ppm (broad,anomeric proton, integral 2.36).

The level of amine substitution was determined by ¹H NMR to be 36% bycalculating the ratio of the sum of the integrals at 1.39-1.65 ppm tothe integral at 4.96 (anomeric protons) and comparing it to the expectedratio for a fully derivatized product (expected ratio of 16:1, asdescribed above).

Preparation of Aminodextran Having an Amine Substitution Level of 13%from Dextran Having a Weight-Average Molecular Weight of 8,500-11,500Daltons (D10K-13 Amine)

Dextran aldehyde (D10K-20) with an oxidation conversion of 20%,equivalent weight per aldehyde group of about 389 Da, was prepared asdescribed above using dextran having a weight-average molecular weightof 8,500-11,500 Da. The dextran aldehyde (5.0 g) was dissolved in 500 mLof 0.1 M borate buffer, pH 11. The dextran aldehyde solution was addedslowly over 5 h using a syringe pump to a basic solution ofhexamethylene diamine (14.9 g, 0.12 mol) dissolved in 296 mL of water.The mixture was stirred at room temperature for 24 h. Then, sodiumborohydride (4.14 g, 0.10 mol) was added, and the reaction mixture wasstirred for 24 h at room temperature. The resulting solution wasfiltered using a TFF device (MWCO 1000, regenerated cellulose membrane).The solution was first concentrated to 1000 mL, then a 12× volume ofwaste was collected, and the resulting solution was lyophilized todryness. The resulting aminodextran is referred to herein as D10K-13amine.

¹H NMR (D₂O): 1.40 ppm, 1.54 ppm, 1.64 ppm (sum of integral of 1.40-1.64ppm: 17.36), 2.3-2.96 ppm (broad, integral 11.16), 3.57-3.93 ppm (broadmultiplet, integral 20.29), 4.98 ppm (broad, anomeric proton, integral2.41).

The level of amine substitution was determined by ¹H NMR to be 13% bycalculating the ratio of the sum of the integrals at 1.40-1.64 ppm tothe integral at 4.98 (anomeric protons) and comparing it to the expectedratio for a fully derivatized product (expected ratio of 16:1, asdescribed above).

Preparation of Aminodextran Having an Amine Substitution Level of 41%from Dextran Having a Weight-Average Molecular Weight of 8,500-11,500Daltons (D10K-41 Amine)

Dextran aldehyde (D10K-50) with an oxidation conversion of 50%,equivalent weight per aldehyde group of about 146 Da, was prepared asdescribed above using dextran having a weight-average molecular weightof 8,500-11,500 Da. The dextran aldehyde (5 g) was dissolved in 500 mLof 0.1 M borate buffer, pH 11.0. The dextran aldehyde solution was addedslowly over 5 hours using a syringe pump to a basic solution ofhexamethylene diamine (36 g) dissolved in 296 mL of deionized water. Themixture was stirred at room temperature for 24 hours, after which timesodium borohydride (4 g) was added, and the reaction mixture was stirredfor another 24 hours. Another aliquot of sodium borohydride (4 g) wasadded, and the reaction mixture was stirred for another 24 hours. Theresulting solution was filtered using a Millipore Pellicon II TFF system(Millipore Corp., Billerica, Mass.) with a 1000 Da MWCO, regeneratedcellulose membrane. The solution was first concentrated to a volume of1000 mL, then a 12× volume of waste was collected, and then theresulting solution was lyophilized to dryness to yield the aminodextran,referred to herein as D10K-41 amine, as a white solid.

¹H NMR: ¹H NMR (D₂O): 1.40 ppm, 1.54 ppm, 1.64 ppm (sum of integral of1.40-1.64 ppm: 11.42), 2.3-2.96 ppm (broad, integral 7.7), 3.57-3.93 ppm(broad multiplet, integral 10.70), 4.98 ppm (broad, anomeric proton,integral 1.72).

The level of amine substitution was determined by ¹H NMR to be 41% bycalculating the ratio of the sum of the integrals at 1.40-1.64 ppm tothe integral at 4.98 (anomeric protons) and comparing it to the expectedratio for a fully derivatized product (expected ratio of 16:1, asdescribed above).

Examples 1-3 Preparation of Hydrogels by Reaction of an Aminodextran anda Dextran Aldehyde

The following Examples demonstrate the preparation of hydrogels byreaction of an aminodextran and a dextran aldehyde. The gel time of thehydrogels was measured.

Into a small vial, 100 μL of an aqueous dextran aldehyde stock solution,as given in Table 1, was added. The vial was tilted and 100 μL of a 50wt % aqueous D10K-46 amine solution was added with care taken not to mixthe two solutions. A timer was started and the two solutions werestirred together with the wooden end of a cotton swab. The initial geltime was defined as the observation of increased viscosity, such that astring formed when the wooden stirring rod was pulled from the gel. Thefinal gel time was defined as the second when stirring pulled the gelfrom the sides of the vial so that the gel could be removed as thewooden stirring rod was pulled from the vial.

The initial and final gel times are given in Table 1. The final geltimes ranged from 10 to 34 sec, depending on the oxidation conversionand the concentration of the dextran aldehyde used.

TABLE 1 Gel Times of Hydrogels Prepared from Aminodextran and DextranAldehyde Dextran Aldehyde Initial Gel Time Final Gel Example Solution(sec) Time (sec) 1 D10K-50 15 21 15 wt % 2 D10K-65 4 10 15 wt % 3D10K-50 28 34 7.5 wt %

Examples 4-6 In Vitro Biocompatibility Testing—Cytotoxicity

The following Examples demonstrate the safety of hydrogels resultingfrom the reaction of an aminodextran with a dextran aldehyde in an invitro test.

The testing was done using NIH3T3 human fibroblast cell culturesaccording to ISO10993-5:1999. The NIH3T3 human fibroblast cells wereobtained from the American Type Culture Collection (ATCC; Manassas, Va.)and were grown in Dulbecco's modified essential medium (DMEM),supplemented with 10% fetal calf serum.

NIH3T3 human fibroblast cell cultures were challenged with hydrogelsmade by combining equal volumes of an aqueous solution of a dextranaldehyde and an aqueous solution of an aminodextran, as shown in Table2. Each hydrogel was placed in the bottom of a well in a polystyreneculture plate such that about ¼ of the well bottoms were covered. Thewells were then sterilized under UV light and seeded with 50,000-100,000NIH3T3 cells.

The cells grew normally confluent and coated the well bottom, growing upto the edges of the hydrogels; however, they did not overgrow thehydrogels. These results, summarized in Table 2, demonstrate a lack ofcytotoxicity of the hydrogels, as well as the lack of adhesion of cellcultures to the hydrogels.

TABLE 2 Cytotoxicity Results Dextran Aldehyde Aminodextran ExampleSolution Solution Cytotoxicity 4 D10K-50 D450K-38 amine nontoxic 20 wt %20 wt % 5 D10K-50 D2000K-36 amine nontoxic 20 wt % 20 wt % 6 D10K-50D60K-32 amine nontoxic 25 wt % 25 wt %

Example 7 In Vitro Biocompatibility Testing—Inflammatory Response

The following Example demonstrate the non-inflammatory response producedby a hydrogel formed by reaction of a dextran aldehyde with anaminodextran in an in vitro test using J774 Macrophage.

The testing was done using J774 Macrophage cultures according toISO10993-5:1999. The J774 Macrophage cells were obtained from ATCC andwere grown in DMEM supplemented with 10% fetal bovine serum.

A J774 mouse peritoneal macrophage cell culture was challenged with ahydrogel made by combining equal volumes of an aqueous solution ofD10K-46 dextran amine (25 wt %) and an aqueous solution of D10K-50dextran aldehyde (20 wt %). The hydrogel was placed on the bottom of awell in a polystyrene culture plate such that about ¼ of the well bottomwas covered. The well was then sterilized under UV light and seeded withJ774 cells. The cell culture was then analyzed for TNF-α, an indicatorof inflammatory response, using an ELISA assay, as described by Lara etal. (Journal of Dental Research 82(6):460-465, 2003). The TNF-α titerwas similar to the negative control (a blank well), indicating thenoninflammatory nature of the hydrogel.

Examples 8-13 In-Vitro Burst Testing of a Sealed Scalpel Incision

The following Examples demonstrate the burst strength of a seal madewith various hydrogels of an incision made in swine uterine horn.

A syringe pump system was used to measure the burst strength of a sealof an incision made in a section of swine uterine horn. The syringe pump(Model No. 22, Harvard Apparatus, Holliston, Mass.) was modified to beequipped with two 30 mL syringes, which were connected together througha “Y” junction. Water was pumped through a single piece of Tygon® R-36tubing (0.6 cm diameter) and through a pressure gauge (Model PDG 5000L,Omega Engineering, Stamford, Conn.).

An approximately 12.5 cm section of clean swine uterine horn, obtainedfrom a local abattoir, was fitted on one end with a metal plug with afeed line fitting for water feed from the syringe pump and on the otherend with a metal plug with a threaded hole which could be sealed with amachine screw. The plugs were held in place with nylon ties around theoutside of the uterine horn. An incision was made through the uterinehorn wall into the interior by puncturing with a Bard Parker™ surgicalblade handle 5 (obtained from BD Surgical Products, Franklin Lakes,N.J.), fitted with a #15 surgical blade. The incision on the outside ofthe uterine horn was wider than the scalpel blade (typically 4-5 mm)while the hole through the inside wall was about 3 mm (about equal tothe blade). This size incision mimics the distance between theinterrupted sutures if an intestine were to be cut and later sutured.The uterine horn was filled with water containing a purple dye via thesyringe pump until water began to leak from the open hole in the endplug and also from the scalpel puncture in the uterine horn wall. Thepump was then turned off and the end plug was sealed with the machinescrew. The scalpel incision site was blotted dry using a paper towel.

The dextran aldehyde and aminodextran solutions were prepared in water.The two solutions were applied to the incision using a double barrelsyringe (Mixpac Systems AG (Rotkreuz, Switzerland) fitted with a 16 stepstatic mixer (Mixpac Systems AG). After the application, the adhesivewas allowed to cure at room temperature for no longer than 2 min.

Burst pressure testing, also referred to herein as leak pressuretesting, was done by pressurizing the sealed uterine horn with waterfrom the syringe pump at a flow rate of 11 mL/min until the bioadhesiveseal began to leak, at which point the pressure was recorded. Adhesivefailure was attributed when the water leaked under the seal between thehydrogel and the tissue surface. Cohesive failure was attributed whenthe water penetrated and leaked through the hydrogel itself. Burstpressure testing was also done on the unsealed uterine horn and the leakpressure was less than 10 mm of mercury (Hg) (less than 1.3 kPa).

The results of the burst testing are summarized in Table 3. The resultsdemonstrate the hydrogels formed by reaction of various dextran aldehydeand aminodextran solutions were able to seal the incision in the swineuterine horn.

TABLE 3 Burst Pressure Testing Results Standard Deviation Dextran AveBurst Burst Aldehyde Aminodextran Pressure, Pressure, Example SolutionSolution mmHg mmHg 8 D10K-50 D60K-32 amine 52.7 17.1 17 wt % 20 wt %(7.0 kPa) (2.3 kPa) 9 D10K-50 D60K-32 amine 45.0 31.8 17 wt % 10 wt %(6.0 kPa) (4.2 kPa) 10 D10K-50 D60K-32 amine 46.9 19.0 10 wt % 20 wt %(6.2 kPa) (2.5 kPa) 11 D10K-50 D10K-46 amine 39.1 13.0 20 wt % 40 wt %(5.2 kPa) (1.7 kPa) 12 D10K-80 D10K-13 amine 64.6 9.3 20 wt % 20 wt %(8.6 kPa) (1.2 kPa) 13 D10K-80 D10K-41 amine 61.5 22.7 20 wt % 40 wt %(8.2 kPa) (3.0 kPa)

Examples 14-16 In Vitro Degradation of Hydrogels

The following Examples demonstrate that the hydrogels formed by reactionof an aminodextran with a dextran aldehyde have low swell and persistfor prolonged periods of time in vitro.

The hydrogel samples were prepared by mixing equal volumes of an aqueoussolution of a dextran aldehyde and an aqueous solution of anaminodextran, as shown in Table 4. After the hydrogels cured, thesamples were weighed and placed inside jars containingphosphate-buffered saline (PBS). The jars were placed inside atemperature-controlled shaker set at 80 rpm and 37° C. The samples wereremoved from the jars at various times, blotted to remove excesssolution, and weighed. Then, the samples were returned to the jars.

The results are summarized in Table 4. The percent swell reported in thetable is the weight of the hydrogel at the specified time divided by theinitial weight of the hydrogel, multiplied by 100. All of the hydrogelswere still present after 13 days (312 h). The results indicate that thehydrogels have low swell and persist for long periods of time.

TABLE 4 Results of In Vitro Degradation of Hydrogels Dextran AldehydeAminodextran % Swell Example Solution Solution 2 h 24 h 120 h 312 h 14D10K-50 D450K-38 amine 41 28 23 9 20 wt % 20 wt % 15 D10K-50 D10K-46amine 106 48 35 27 20 wt % 20 wt % 16 D10K-50 D60K-32 amine 98 44 29 2325 wt % 20 wt %

Example 17 In Vitro Testing of Dextran Amine for Inhibition of E. coliGrowth

The following Example demonstrates the antimicrobial activity of dextranamine by testing inhibition of E. coli growth.

A suspension culture of E. coli (strain K12, ATCC No. 25257) fromAmerican Type Culture Collection (ATCC; Manassas, Va.) was prepared byseeding an individual colony into 4 mL of Luria Broth (obtained fromATCC). The culture was incubated in a shaker overnight at 37° C. toallow the culture to reach the saturation point of cell growth. Theconcentration of E. coli in the saturated overnight culture wasapproximately 1×10⁹ cells/mL. The saturated overnight culture (10 μL)was then seeded into 4 mL of Luria Broth, and the desired amount of adextran amine was added, as indicated in Table 5. The culture wasincubated in a shaker overnight at 37° C. Following the incubation,bacterial growth was quantified by measuring the optical density of theculture at 600 nm using a spectrophotometer. This method for determiningin vitro antimicrobial activity has been shown to correlate with in vivoantimicrobial activity (Lee S H et al., Journal of Pharmacy andPharmacology 2003 April; 55:559-66). The results are summarized in Table5. The bacteriostatically effective amount of dextran amine, i.e., theamount of the dextran amine that produced a 0.5 log decrease inbacterial growth, is also given in the Table. The bacteriostaticallyeffective amount of the dextran amine was estimated from a plot of thelog of bacterial growth (cells/mL) versus the volume of dextran aminesolution added to the culture medium.

TABLE 5 Inhibition of E. coli Growth by Dextran Amines Volume BacterialBacteriostatically Dextran Added Growth Effective Example amine (μL)(cells/mL) Amount (mg/mL) 17 D10K-41 0 1.0 × 10⁹ ≧5 amine 20 7.1 × 10⁸10 wt % 50 1.7 × 10⁸

1. A kit comprising: a) at least one dextran that has been derivatizedto provide at least one aminodextran that contains primary amine groups,said at least one dextran having a weight-average molecular weight ofabout 1,000 to about 1,000,000 Daltons, said at least one aminodextranhaving an amine substitution level of about 5% to about 65%; and b) atleast one dextran that has been oxidized to provide at least oneoxidized dextran containing aldehyde groups, said at least one dextranhaving a weight-average molecular weight of about 1,000 to about1,000,000 Daltons, said at least one oxidized dextran having anequivalent weight per aldehyde group of about 65 to about 1500 Daltons;wherein the at least one aminodextran and the at least one oxidizeddextran are unreacted.
 2. The kit according to claim 1 wherein theaminodextran is a first aqueous solution or dispersion and the oxidizeddextran a second aqueous solution or dispersion.
 3. The kit according toclaim 2 wherein the first aqueous solution or dispersion contains theaminodextran at a concentration of about 5% to about 70% by weightrelative to the total weight of the solution or dispersion.
 4. The kitaccording to claim 2 wherein the second aqueous solution or dispersioncontains the oxidized dextran at a concentration of about 5% to about50% by weight relative to the total weight of the solution ordispersion.
 5. The kit according to claim 1 wherein the aminodextran andthe oxidized dextran are finely divided powders.
 6. The kit according toclaim 1 wherein the equivalent weight per aldehyde group of the oxidizeddextran is about 90 to about 1500 Daltons.
 7. A dried hydrogel productformed by a process comprising the steps of: a) reacting in a solvent(i) at least one dextran that has been derivatized to provide at leastone aminodextran that contains primary amine groups, said at least onedextran having a weight-average molecular weight of about 1,000 to about1,000,000 Daltons, said at least one aminodextran having an aminesubstitution level of about 5% to about 65%; with (ii) at least onedextran that has been oxidized to provide at least one oxidized dextrancontaining aldehyde groups, said at least one dextran having aweight-average molecular weight of about 1,000 to about 1,000,000Daltons, said at least one oxidized dextran having an equivalent weightper aldehyde group of about 65 to about 1500 Daltons, to form ahydrogel; b) treating said hydrogel to remove at least a portion of saidsolvent to form the dried hydrogel.
 8. The dried hydrogel according toclaim 7, wherein said dried hydrogel is a film.
 9. The dried hydrogelaccording to claim 7 wherein the process further comprises comminutingthe dried hydrogel to form finely divided particles.
 10. The driedhydrogel according to claim 7, wherein the equivalent weight peraldehyde group of the oxidized dextran is about 90 to about 1500Daltons.
 11. A composition formed by the combination of a) at least onedextran that has been derivatized to provide at least one aminodextranthat contains primary amine groups, said at least one dextran having aweight-average molecular weight of about 1,000 to about 1,000,000Daltons, said at least one aminodextran having an amine substitutionlevel of about 5% to about 65%; with b) at least one dextran that hasbeen oxidized to provide at least one oxidized dextran containingaldehyde groups, said at least one dextran having a weight-averagemolecular weight of about 1,000 to about 1,000,000 Daltons, said atleast one oxidized dextran having an equivalent weight per aldehydegroup of about 65 to about 1500 Daltons.
 12. The composition accordingto claim 11, wherein the aminodextran is a first aqueous solution ordispersion and the oxidized dextran is a second aqueous solution ordispersion.
 13. The composition according to claim 12, wherein the firstaqueous solution or dispersion contains the aminodextran at aconcentration of about 5% to about 70% by weight relative to a totalweight of the solution or dispersion.
 14. The composition according toclaim 12, wherein the second aqueous solution or dispersion contains theoxidized dextran at a concentration of about 5% to about 50% by weightrelative to the total weight of the solution or dispersion.
 15. Thecomposition according to claim 11, wherein the aminodextran and theoxidized dextran are finely divided powders.
 16. The compositionaccording to claim 11, wherein the equivalent weight per aldehyde groupof the oxidized dextran is about 90 to about 1500 Daltons. 17.(canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)22. (canceled)
 23. A product comprising the dried hydrogel of claim 7.24. The product according to claim 23, wherein said dried hydrogel is afilm.
 25. The product according to claim 23, wherein said dried hydrogelis finely divided particles.